AUTHOR=Zhang Dai , Qiang Ran , Zhao Jing , Zhang Jinglin , Cheng Jianing , Zhao Dongmei , Fan Yaning , Yang Zhihui , Zhu Jiehua 

TITLE=Mechanism of a Volatile Organic Compound (6-Methyl-2-Heptanone) Emitted From Bacillus subtilis ZD01 Against Alternaria solani in Potato

JOURNAL=Frontiers in Microbiology

VOLUME=12

YEAR=2022

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2021.808337

DOI=10.3389/fmicb.2021.808337

ISSN=1664-302X

ABSTRACT=<p>The antagonistic mechanisms of soluble non-volatile bioactive compounds, such as proteins and lipopeptides emitted from <italic>Bacillus</italic> have been widely studied. However, there are limited studies on the antifungal mechanisms of volatile organic compounds (VOCs) produced by <italic>Bacillus</italic> against plant fungal diseases. In this study, the antagonistic mechanisms of one specific VOC, 6-methyl-2-heptanone, against <italic>Alternaria solani</italic> were investigated. To optimize the extraction conditions of headspace solid-phase microextraction, a 50/30-μm divinylbenzene/carboxen/polydimethylsiloxane fiber at 50°C for 40 min was used. For gas chromatography-mass spectrometry using a free fatty acid phase capillary column, 6-methyl-2-heptanone accounted for the highest content, at 22.27%, of the total VOCs from <italic>Bacillus subtilis</italic> ZD01, which inhibited <italic>A. solani</italic> mycelial growth strongly <italic>in vitro</italic>. Therefore, 6-methyl-2-heptanone was selected as the main active chemical to elucidate the action mechanisms against <italic>A. solani</italic>. Scanning and transmission electron microscopy analyses revealed that after exposure to an EC<sub>50</sub> dose of 6-methyl-2-heptanone, <italic>A. solani</italic> hyphal cells had a wide range of abnormalities. 6-Methyl-2-heptanone also caused the capture of cellular fluorescent green label and the release of adenosine triphosphate (ATP) from outer membranes <italic>A. solani</italic> cells, which may enhance 6-methyl-2-heptanone ability to reach the cytoplasmic membrane. In addition, 6-methyl-2-heptanone showed strong inhibitory effect on <italic>A. solani</italic> conidial germination. It also damaged conidial internal structures, with the treated group having collapsed shrunken small vesicles as observed by transmission electron microscopy. Because 6-methyl-2-heptanone showed strong effects on mycelial integrity and conidial structure, the expression levels of related pathogenic genes in <italic>A. solani</italic> treated with 6-methyl-2-heptanone were investigated. The qRT-PCR results showed that transcriptional expression levels of <italic>slt2</italic> and <italic>wetA</italic> genes were strongly down-regulated after exposure to 6-methyl-2-heptanone. Finally, because identifying the functions of pathogenic genes will be important for the biological control of <italic>A. solani</italic>, the <italic>wetA</italic> gene was identified as a conidia-associated gene that plays roles in regulating sporulation yield and conidial maturation. These findings provide further insights into the mechanisms of VOCs secreted by <italic>Bacillus</italic> against <italic>A. solani</italic>.</p>